1. Field of the Invention
The present invention relates to a marker for detecting and measuring free radical damage; a method for the direct detection and measurement of the damaging activity of free radicals in vivo is provided.
2. Discussion of the Background
Free radicals are atoms or groups of atoms with an odd (unpaired) number of electrons and can be formed when oxygen interacts with certain molecules. Once formed these highly reactive radicals can start a chain reaction, like dominoes. Their chief danger comes from the damage they can do when they react with important cellular components such as DNA, or the cell membrane. Cells may function poorly or die if this occurs.
A free radical is any molecular species capable of independent existence, that contains one or more unpaired valence electrons not contributing to intramolecular bonding, and is—in that sense—“free”. Free radicals are produced by oxidation/reduction reactions in which there is a transfer of only one electron at a time, or when a covalent bond is broken and one electron from each pair remains with each atom. Free radicals are highly reactive, owing to the tendency of electrons to pair—that is, to pair by the receipt of an electron from an appropriate donor or to donate an electron to an appropriate acceptor. Thus, once formed, free radicals initiate a chain reaction, like dominoes—whenever a free radical reacts with a non-radical, a chain reaction is initiated until two free radicals react and then terminate the propagation with a 2-electron bond (each radical contributing its single unpaired electron).
In biological systems free radicals have a range of transitory existences depending upon their reactivity. Some are stable, e.g. melanins can have a long lifetime, moderately stable ones such as nitric oxide can have lifetimes of ˜5 seconds and highly unstable ones such as hydroxyl radicals exist for only a hundredth of a microsecond. The chief danger of free radicals is the damage they can do when they react with important cellular components such as DNA, or the cell membrane. The result on cells of action by free radicals can be diminished or impaired cellular functioning, or even death. For instance, oxygen-free radicals are believed to play a significant role in the aging process. These free radicals often take an electron away from a “target” molecule to pair with their single free electron. This process is referred to as “oxidation” and is a known cause of cellular damage and death. Oxygen-free radicals are also implicated in many diseases including neurodegenerative diseases (ALS, Parkinson's, Alzheimer's), cataractogenesis, atherosclerosis, diabetes mellitus, ischemia-reperfusion injury, kwashiorkor, and certain toxicities, to mention only a few.
There are many sources of free radicals both within and external to cells. Free radicals are produced by normal ongoing metabolism, especially from the electron transport system in the mitochondria and from a number of normally functioning enzymes. Examples of naturally produced free radicals are: xanthine oxidase, cytochrome p450, monoamine oxidase, and nitric oxide synthase. In the brain, free radicals are produced from the autoxidation of norepinephrine and dopamine. The autoxidation of catechols to quinones generates reduced forms of molecular oxygen, sources of free radicals (e.g., superoxide and hydrogen peroxide). One study suggests that oxidants generated by mitochondria are the major source of oxidative lesions that accumulate with age. See Ames, B. N., M. K. Shigenaga and T. M. Hagen, “Oxidants, antioxidants, and the degenerative diseases of aging.” Proc. Natl. Acad Sci. USA 90: 7915-7922 (1993).
Free radicals also function beneficially in normal physiology, including information processing in the brain. Since free radicals can donate an electron to an appropriate acceptor (“reduction reaction”) or pair their unpaired electron by taking one from an appropriate donor (“oxidation reaction”) they have major influences on the so-called “redox state” in cells—important in normal physiological regulatory reactions. Major free radical targets are molecular complexes that readily give up or acquire a single electron, e.g., those with sulfhydryl/disulfides or with paramagnetic metals (iron, copper). It is calculated that endogenously generated oxygen free radicals make about 10,000 oxidative interactions with DNA per human cell per day (Ames et al., 1993, supra).
Under normal conditions the damaging actions of oxygen free radicals are minimized by abundant protective and repair mechanisms that cells possess, including many enzymes (e.g. superoxide dismutase, catalase) and redox active molecules (e.g., glutathione, thioredoxin).
There is currently an overwhelming need for a sensitive test of free radical damage. For instance, it has been found that the DNA in breast cancer tumors that have generated metastases contain more than twice the amount of free radical damage than tumors that remained confined to the breast. The ability to detect this free radical damage would allow identification of an identifiable metastatic pattern or “profile,” which would be of great benefit in determining if newly diagnosed breast cancer was likely to spread and whether aggressive radiation and chemotherapy is needed. See, e.g., Malins D. et al., “Progression of Human Breast Cancers to the Metastatic State Is Linked to Hydroxyl Radical-induced DNA Damage,” Proc. Natl. Acad. Sci. USA 93:2557-2563 (1996). However, no viable test exists.
There currently exists several limited methods for detection of the damaging activity of free radicals in the body. One method uses an isoprostane called IPF2alpha-I, an abundant and stable byproduct of free-radical catalyzed oxidation of arachidonic acid, which is easily detected in urine. Arachidonic acid is a fatty molecule found in cell membranes throughout the body. Pratico D. et al., “Localization of distinct F2-isoprostanes in human atherosclerotic lesions,” J. Clin. Invest. 100(8):2028-2034 (1997). Other methods take advantage of the formation of carbonyls from lipids, proteins, carbohydrates, and nucleic acids during oxidative stress. For example, metal-catalyzed, “site-specific” oxidation of several amino acid side-chains has been reported to result in the production of aldehydes or ketones, and peroxidation of lipids to generate reactive aldehydes such as malondialdehyde and hydroxynonenal. These oxidative changes have been detected in situ using 2,4-dinitrophenylhydrazine labeling linked to an antibody system specific to localized biomacromolecule-bound carbonyl reactivity. See Smith, M. A. et al., “Cytochemical demonstration of oxidative damage in Alzheimer disease by immunochemical enhancement of the carbonyl reaction with 2,4-dinitrophenylhydrazine,” J. Histochem. Cytochem. 46(6):731-735 (1998). Use of immunochemical assays for detection of carbonyl moieties resulting from oxidative damage to bovine serum albumin has also been reported. See Keller, R. J. et al., “Immunochemical detection of oxidized proteins,” Chem. Res. Toxicol. 6(4): 430-433 (1993), and Mateos-Nevado, D. J., “Immunological detection and quantification of oxidized proteins by labeling with digoxigenin,” Biosci. Biotechnol. Biochem. 62(3):419-423 (1998).
These methods, however, are limited in their usefulness and applicability due to the highly specific and system-limited nature of the markers utilized for detection. The present invention, in contrast, provides a marker for the existence and detection/measurement of free radical damage which is highly sensitive and present in a majority of human fluids and tissues.